Clathrin-coated vesicles mediate selective uptake of receptors from the plasma membrane. They are responsible for rapid down-regulation of hormone and signaling receptors, and for efficient endocytosis of recycling receptors that bind nutrients and proteins destined for degradation. Clathrin-coated vesicles also participate in transport of proteins from the trans-Golgi network (TGN) to lysosomes and in maturation of secretory granules. The clathrin coat is a polyhedral protein network that forms on membranes by self-assembly of soluble clathrin molecules from the cytoplasm. Polymerization of clathrin onto membranes is controlled by adaptor molecules which comprise the second major protein component of the clathrin coat. incorporation of receptors into a clathrin-coated vesicle is selective and involves recognition of their cytoplasmic domains by the adaptor molecules. There are two types of adaptor molecules, each involved in nucleating clathrin assembly at a defined intracellular location. The AP2 adaptor controls formation of endocytic coated vesicles and the AP1 adaptor controls formation of coated vesicles in the TGN. in addition to adaptors, other intracellular factors have been implicated in the regulation of coated vesicle formation, particularly during specialized functions. The goal of this proposal is to define the molecular basis for control of clathrin-coated vesicle formation within cells. The first specific aim is to identify the targets for localization of clathrin assembly by adaptor molecules. To this end, plasma membrane and TGN membrane proteins-that bind to affinity columns of purified AP1 and AP2 will be characterized. In addition, monoclonal antibodies will be produced against proteins present in the target membranes and will be screened for their ability to inhibit adaptor-membrane interactions. The second specific aim is to determine the subdomains of adaptor subunits responsible for their defined functions in clathrin assembly localization, receptor recognition and endosome formation. Adaptor subunits and fragments thereof will be produced using biochemical and molecular genetic procedures and will be tested for activity in in vitro assays to map functional domains. The third specific aim is to characterize the role of extrinsic factors in clathrin-coated vesicle formation. This will involve characterization of a factor that is required for clathrin binding to adaptors during reconstitution of clathrin assembly onto plasma membrane fragments, in vitro. In addition, the patterns of phosphorylation of clathrin and adaptor subunits during specialized clathrin-mediated functions will be established, and the function of the neuron-specific assembly protein AP180 will be investigated. These studies will result in better understanding of the molecular recognition events which control clathrin- mediated membrane transport and will contribute to manipulation of these processes in the design of receptor-mediated therapeutics and vaccine production.